The broad, long-term objective of the project is to understand the importance of calcium (Ca2+) signaling in pathogenesis offamilial Alzheimer's disease (FAD), Presenilins are transmembrane proteins localized to endoplasmic reticulum (ER), Missense mutations in presenilin-1 (PS1) and presenilin-2 (PS2) account for 40% of FAD cases. Presenilins function as catalytic subunit of gamma-secretase which cleaves amyloid precursor protein (APP) and releases A-beta peptide, a principal component of amyloid plaques in AD brains. We recently discovered that presenilins also function as passive ER Ca2+ leak channels which control intraluminal ER Ca2+ concentration. Moreover, we found that many FAD-linked mutations in presenilins impair their ER Ca2+ leak channel function. Here I propose to further investigate the effects of FAD mutations in presenilins on their ER Ca2+ leak function in the context of AD pathology. Specifically, I propose: 1. To analyze effects of additional FAD mutations in presenilin-1 on ER Ca2+ leak function. The experiments with primary Iymphoblasts established from FAD patients and rescue experiments with PS double-knockout (DKO) mouse embryonic fibroblasts (MEF) will be performed. The main goal of these experiments will be to establish a correlation between clinical PS-FAD phenotypes with ER Ca2+ leak function of PS-FAD mutants. Obtained results will help to explain a variability of clinical phenotypes resulting from PS-FAD mutations. The results obtained in Ca2+ imaging experiments will be correlated with effects of the same PS-FAD mutations on Abeta42 and Abeta40 production measured by immunoassay. 2. To map the ion conduction pathway in PS1 by cysteine-scanning mutagenesis. I propose that the ion conduction pore of presenilin channels is formed by transmembrane domains 6, 7 and 9. To test this hypothesis, Cys point mutants will be introduced in transmembrane domains 6, 7 and 9 of mouse cysteine-less PS1 construct. The function of generated mPS1-Cys mutants will be evaluated in PS DKO MEF rescue experiments. The functional mPS1-Cys mutants will be expressed in Sf9 cells by baculoviral infection and reconstituted into planar lipid bilayers (BLM). The surface accessibility of introduced cysteine residues will be tested by applying thiol-specific modifying methanethiosulfonate (MTS) reagents (MTSEA, MTSET and MTSES) to the BLM. Results of these experiments will provide mechanistic information about critical determinants responsible for ion-conduction properties of presenilins. The results of proposed experiments will help to further establish the connection between FAD mutations in presenilins, disturbances in ER Ca2+ signaling and AD pathogenesis. These data will also help to evaluate "Ca2+ hypothesis of AD" and will contribute to selecting optimal strategies for treatment of AD.